Optical microdisks for integrated devices

ABSTRACT

Apparatus and methods for improving optical signal collection in an integrated device are described. A microdisk can be formed in an integrated device and increase collection and/or concentration of radiation incident on the microdisk and re-radiated by the microdisk. An example integrated device that can include a microdisk may be used for analyte detection and/or analysis. Such an integrated device may include a plurality of pixels, each having a reaction chamber for receiving a sample to be analyzed, an optical microdisk, and an optical sensor configured to detect optical emission from the reaction chamber. The microdisk can comprise a dielectric material having a first index of refraction that is embedded in one or more surrounding materials having one or more different refractive index values.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.62/884,395, filed Aug. 8, 2019 and titled “Optical Microdisks forIntegrated Devices,” which is herein incorporated by reference in itsentirety.

FIELD OF THE APPLICATION

The present application relates to optical microdiskS that can increasecollection efficiency and/or concentration of radiation within anintegrated device.

BACKGROUND

Some microfabricated chips may be used to analyze a large number ofanalytes or specimens in parallel. In some cases, optical excitationradiation is delivered to a plurality of discrete sites on a chip atwhich separate analyses are performed. The excitation radiation mayexcite a specimen at each site, a fluorophore attached to the specimen,or a fluorophore involved in an interaction with the specimen. Inresponse to the excitation, radiation may be emitted from a site and theradiation can be detected by a sensor. Information obtained from theemitted radiation for a site, or lack of emitted radiation, can be usedto determine a characteristic of the specimen at that site.

SUMMARY

Apparatus and methods relating to optical microdisks are described. Suchoptical microdisks may improve collection and/or concentration ofradiation incident on the microdisk and passing through the microdisk toa detector. Optical microdisks may be formed in integrated devices thatinclude optical sensors, such as photodiodes, CCD photodiode arrays,CMOS photodiode arrays, image sensor arrays, fluorescent sensor arrays,bio-sensor chips, and integrated devices (or lab on chips) adapted forgenetic sequencing and/or protein sequencing, for example. In suchapplications, radiation to be detected may be very low in intensity,leading to a small signal-to-noise ratio (SNR) and decreased sensingaccuracy. Including an optical microdisk in such a device may help focusor concentrate the radiation onto a sensor, thereby increasing the SNR,which can result in increased sensing accuracy and/or faster sensing.

In an example embodiment, an optical microdisk can be used in connectionwith instruments for analyzing specimens, where optical detection isused to analyze the radiation emitted by a specimen or fluorophoreattached to or associated with a specimen in response to opticalexcitation delivered to the specimen. Specimens may include biologicalmaterials such as genetic material or proteins that are to be analyzedby the instrument. More generally, embodiments of optical microdisksdescribed herein may be used in applications in which it is desired toincrease SNR by increasing the collection and/or concentration ofemission radiation or other radiation (such as for imaging, display, oroptical communication purposes). Among other possible contexts, opticalmicrodisks described herein may be used in conjunction with, forexample, integrated detectors in optical communication systems, LEDemitter arrays, and/or imaging arrays.

Some embodiments relate to a microfabricated structure on a substratehaving one or more pixels. Each pixel may comprise a reaction chamber, awaveguide configured to deliver excitation radiation to the reactionchamber, an optical sensor configured to detect emission radiationemitted from the reaction chamber, and a microdisk disposed between thewaveguide and the optical sensor and configured to increase an amount ofthe emission radiation that is received by the optical sensor comparedto an amount of the emission radiation that would be received by theoptical sensor without the microdisk.

Some embodiments relate to a method of operating an integrated device.The method includes acts of: delivering excitation energy from anoptical waveguide to a reaction chamber, wherein the optical waveguideand reaction chamber are integrated on a substrate of the integrateddevice; passing emission radiation from the reaction chamber through amicrodisk to a sensor that is integrated on the substrate; andincreasing with the microdisk an amount of the emission radiationreceived by the sensor compared to an amount of the emission radiationthat would be received without the microdisk.

Some embodiments relate to a method of fabricating an integrated device.The method includes acts of: forming, at each of a plurality of pixelson a substrate, a reaction chamber, an optical waveguide arranged todeliver excitation radiation to the reaction chamber, and an opticalsensor arranged to receive emission radiation from the reaction chamber;and forming a microdisk at each pixel between the waveguide and theoptical sensor, wherein the microdisk is configured to increase anamount of the emission radiation that is received by the optical sensorcompared to an amount of the emission radiation that would be receivedwithout the microdisk.

The foregoing apparatus and method embodiments may be implemented withany suitable combination of aspects, features, and acts described aboveor in further detail below. These and other aspects, embodiments, andfeatures of the present teachings can be more fully understood from thefollowing description in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

Various aspects and embodiments will be described with reference to thefollowing figures. It should be appreciated that the figures are notnecessarily drawn to scale. In the drawings, each identical or nearlyidentical component that is illustrated in various figures isrepresented by a like numeral. For purposes of clarity, not everycomponent may be labeled in every drawing.

FIG. 1-1 depicts an example of a portion of an integrated device havingan array of reaction chambers that can be excited optical pulses via oneor more waveguides and corresponding detectors for each reactionchamber, according to some embodiments;

FIG. 1-2 depicts further details of a pixel comprising a reactionchamber, optical waveguide, and sensor, according to some embodiments.

FIG. 1-3 depicts an example of a biological reaction that can occurwithin a reaction chamber, according to some embodiments.

FIG. 1-4 depicts an example of polypeptide sequencing that can occurwithin a reaction chamber, according to some embodiments.

FIG. 1-5 depicts an example of polypeptide sequencing that can occurwithin a reaction chamber, according to some embodiments

FIG. 1-6 schematically depicts an example microfabricated structure thatincludes a microdisk at a pixel of an integrated device, according tosome embodiments;

FIG. 1-7 schematically depicts an example microfabricated structure thatincludes a microdisk surrounded by rings at a pixel of an integrateddevice, according to some embodiments;

FIG. 2-1 schematically depicts an example microfabricated structure at apixel of an integrated device, according to some embodiments;

FIG. 2-2 depicts a computer-simulated optical emission pattern from areaction chamber at a pixel of an integrated device, according to someembodiments;

FIG. 2-3 depicts a computer-simulated optical emission pattern from areaction chamber at a pixel of an integrated device, according to someembodiments;

FIG. 3-1 is a plot illustrating normalized emission collectionefficiency as a function of the location of a microdisk in an integrateddevice, according to some embodiments;

FIG. 3-2 is a plot illustrating emission collection efficiency with andwithout a microdisk as a function of iris diameter, according to someembodiments;

FIG. 3-3 is a contour plot illustrating normalized collection efficiencyas a function of microdisk diameter and iris diameters, according tosome embodiments;

FIG. 3-4 is a contour plot illustrating an increase in an amount ofemission radiation received by a sensor due to the microdisk as afunction of upper and lower iris diameters, according to someembodiments;

FIG. 4-1 is a scanning electron microscope image of an examplemicrofabricated structure at a pixel of an integrated device, accordingto some embodiments;

FIG. 4-2 is a scanning electron microscope image of an examplemicrofabricated structure at a pixel of an integrated device, accordingto some embodiments;

and

FIG. 5-1A, FIG. 5-1B, FIG. 5-1C, FIG. 5-1D, FIGS. 5-1E, and 5-1F depictstructures associated with an example method for fabricating amicrodisk, according to some embodiments.

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings. When describing embodiments in referenceto the drawings, directional references (“above,” “below,” “top,”“bottom,” “left,” “right,” “horizontal,” “vertical,” etc.) may be used.Such references are intended merely as an aid to the reader viewing thedrawings in a normal orientation. These directional references are notintended to describe a preferred or only orientation of features of anembodied device. A device may be embodied using other orientations.

DETAILED DESCRIPTION I. Integrated Device with an Optical Microdisk

The inventors have recognized and appreciated that a compact, high-speedapparatus for performing detection of single molecules, proteins, orparticles could reduce the cost of performing complex quantitativemeasurements of biological and/or chemical samples and rapidly advancethe rate of biochemical technology discoveries. Moreover, acost-effective device that is readily transportable could provide peoplein developing regions access to essential diagnostic tests that coulddramatically improve their health and well-being. For example,embodiments described herein may be used for diagnostic tests of blood,urine and/or saliva that may be used by individuals in their home, or bya doctor at a mobile clinic or a remote clinic in a developing country.In some cases, it is desirable to have portable, hand-held instrumentsfor analyzing samples, so that technicians or medical personnel caneasily carry the instrument into the field where service is needed andanalyze a sample quickly and accurately. According to some embodiments,a portable instrument may be used for gene sequencing, proteinsequencing, or performing conventional sample analyses, such as completeblood count analysis. In more advanced clinical settings, a desk-topsize instrument may be desired for more complex sample analyses.

Instruments for analyzing such samples may utilize microfabricatedstructures and devices (e.g., electronic amplifiers, logic devices,optoelectronic devices, and/or microfluidic devices, etc.) that areformed on one or more chips. Such chips can help reduce the overall sizeof the instrument. Dies for such chips may have one or a plurality(e.g., hundreds, thousands, millions, or more) of pixels, eachcomprising one or more microfabricated devices configured to participatein analyte detection and/or signal analysis. In some implementations, apackaged die (also referred to as an “integrated device” or “chip”herein) may be a single-use, disposable element that a user inserts intoan instrument for a measurement and disposes after completion of themeasurement. In some cases, the instrument and integrated device may beconfigured for biomolecular detection and/or analysis. The molecules maybe, by way of example and not limitation, proteins and/or DNA. Such anintegrated device may be used to perform massively parallel analyses ofspecimens (e.g., perform “biological assays” or “bioassays”), therebyincreasing the speed at which such biological analyses may be completed.In some embodiments, the disposable integrated device may be mountedinto a receptacle of an advanced analytic instrument by a user, andinterface with optical and electronic components in the instrument. Thedisposable integrated device can be replaced easily by a user for eachnew sample analysis.

In some embodiments, an integrated device can comprise abio-optoelectronic chip on which a large number of pixels havingreaction chambers are formed and arranged for parallel optical analysesof analytes. An example portion of a bio-optoelectronic chip is depictedin FIG. 1-1, which shows reaction chambers 1-130 and correspondingsensors 1-122 for each of a plurality of pixels (eight, in thisexample). When an analyte is present in a reaction chamber 1-130 andtagged with one or a plurality of luminescent markers (also referred toherein as “fluorophores”), the marker(s) may be excited by excitationradiation 1-121 delivered via an optical waveguide 1-112 to the reactionchamber 1-130. Emission radiation from the marker(s) can be detected bya corresponding sensor 1-122 and used to identify the type of marker(s)that is(are) present in pixel, which in turn can provide informationabout the analyte in the pixel. The inventors have recognized andappreciated that an analysis based on detection of emission radiationfrom the marker(s) may be significantly affected by the signal-to-noiseratio (SNR), since the amount of emission radiation (signal) from one ormore markers in a pixel can be very low. Sources of optical noise caninclude any source of optical radiation that is not from the opticalemission of the luminescent marker(s) in a reaction chamber 1-130 of acorresponding sensor 1-122 (e.g., stray emission radiation from anadjacent reaction chamber, scattered excitation radiation from regionsof the waveguide 1-112, 1-115, scattered light from outside the chip,etc.). By increasing the amount of emission radiation from a reactionchamber 1-130 that reaches a respective optical sensor 1-122 within thepixel, the SNR may be increased resulting in faster and/or more accuratemeasurements.

One way to increase the amount of emission radiation from a reactionchamber 1-130 that is received by a corresponding optical sensor 1-122is to locate an optical microdisk 1-605 between the reaction chamber1-130 and sensor 1-122, as in an example embodiment depicted in FIG.1-4. An optical microdisk formed from a dielectric material can collectemission radiation from the reaction chamber 1-130 and redirect theemission radiation to the optical sensor 1-122 that might otherwise belost. Embodiments of an optical microdisk collector (hereinafter,“optical microdisk” or “microdisk”) for increasing the SNR in integratedoptical sensing applications are described below. It should beappreciated that various aspects relating to an optical microdisk thatare described herein may be implemented in any of numerous ways.Examples of specific implementations are provided herein forillustrative purposes only. In addition, the various aspects describedin the embodiments below may be used alone or in any combination, andare not limited to the combinations explicitly described herein.

Returning to FIG. 1-1, in some embodiments, the excitation radiation1-121 may be coupled to one or more waveguides 1-112 via a gratingcoupler 1-110, though coupling to an end of an optical waveguide may beused in some cases. Excitation radiation 121 may be generated by aradiation source such as is described in U.S. Patent Publication No.2015/0141267, filed on Nov. 17, 2014 and titled “Integrated Device withExternal Light Source for Probing Detecting and Analyzing Molecules,”which is incorporated herein by reference in its entirety. According tosome embodiments, a quad detector 1-120 may be located on asemiconductor substrate 1-105 (e.g., a silicon substrate, though othersemiconductor materials may be used) near the grating coupler 1-110 foraiding in alignment of the beam of excitation radiation 1-121 to thegrating coupler 1-110. In some implementations, one or more sensors1-122 may be used to sense excitation radiation and aid in alignment ofthe excitation radiation 1-121 to the grating coupler 1-110. The sensors1-122 may comprise photodetectors (e.g., time-binning photodetectors orsingle-photon avalanche photodiodes). The one or more waveguides 1-112and reaction chambers 1-130 may be integrated on the same semiconductorsubstrate 1-105 with intervening dielectric layers (e.g., silicondioxide layers, not shown) between the substrate, waveguide, reactionchambers, and sensors 1-122. The sensor 1-122 may connect to othercomplementary metal-oxide-semiconductor (CMOS) circuitry on thesubstrate via interconnects (not shown). A distance from the bottom ofthe optical waveguide 1-115 to the sensor 1-122 can be between 500 nmand 10 μm.

Each waveguide 1-112 may include a tapered portion 1-115 below thereaction chambers 1-130 to equalize optical power coupled to thereaction chambers along the waveguide. The reducing taper may force moreexcitation radiation energy outside the waveguide's core, increasingcoupling to the reaction chambers and compensating for optical lossesalong the waveguide, including losses for excitation radiation couplinginto the reaction chambers. A second grating coupler 1-117 may belocated at an end of each waveguide to direct optical energy to anintegrated photodiode 1-124. The integrated photodiode may detect anamount of power coupled down a waveguide and provide an electricalsignal to feedback circuitry that controls, for example, a beam-steeringmodule that controls the position and angle of excitation radiation1-121 incident on the grating coupler 1-110, for example.

The reaction chambers 1-130 may be aligned with the tapered portion1-115 of the waveguide and recessed in a tub 1-140. A metal coatingand/or multilayer coating 1-150 may be formed around the reactionchambers and above the waveguide to prevent excitation of fluorophoresthat are not in the reaction chambers 1-130 (e.g., dispersed in asolution above the reaction chambers). The metal coating and/ormultilayer coating 1-150 may be raised beyond edges of the tub 1-140 toreduce absorptive losses of the excitation energy in the waveguide 1-112at the input and output ends of each waveguide. In some implementations,a multilayer, discriminating optical structure may be formed above eachsensor 1-122 and configured to preferentially attenuate excitationradiation over emission from the fluorophores.

In some embodiments, a reaction chamber 1-130 may be formed in atransparent or semitransparent material, such as an oxide or a nitride,so that excitation radiation from the optical waveguide 1-115 andemission radiation from the reaction chamber 1-130 may pass through thetransparent or semitransparent material without being attenuated by morethan 10%, for example. The reaction chambers 1-130 may have a depthbetween 50 nm and 1 μm, according to some embodiments. A minimumdiameter of a reaction chamber 1-130 may be between 50 nm and 300 nm insome embodiments. If a reaction chamber 1-130 is formed as a zero-modewaveguide, then the minimum diameter may be even less than 50 nm in someimplementations. If large analytes are to be analyzed, the minimumdiameter may be larger than 300 nm. The reaction chamber may be locatedabove the optical waveguide 1-115 such that a bottom of the reactionchamber may be up to 500 nm above a top of the waveguide 1-115.

There may be a plurality of rows of waveguides 1-112, reaction chambers1-130, and photodetectors 1-122 on the integrated device in addition tothe single row shown in FIG. 1-1. For example, there may be 64 rows,each having 512 reaction chambers, for a total of 32,768 reactionchambers in some implementations. Other implementations may includefewer or more reaction chambers, and may include other layoutconfigurations. In some cases, there can be more than 64 rows and morethan 512 reaction chambers in a row, such that the total number ofpixels and reaction chambers on a chip can be between 64,000 and10,000,000. Excitation radiation power may be distributed to themultiple waveguides 1-112 via one or more star couplers or multi-modeinterference couplers (not shown), or by any other means, locatedbetween an optical coupler 1-110 and the plurality of waveguides 1-112.In some cases, an optical coupler 1-110 may span a plurality ofsingle-mode waveguides 1-112, such that an input beam is coupledsimultaneously into the plurality of single-mode waveguides 1-112, asdescribed in U.S. Provisional Patent Application No. 62/861,832, filedJun. 14, 2019, titled “Sliced Grating Coupler with Increased BeamAlignment Sensitivity,” which application is incorporated by referenceherein in its entirety. Waveguides 1-112 and reaction chambers 1-130 canbe formed by microfabrication processes described in U.S. patentapplication Ser. No. 14/821,688, filed Aug. 7, 2015, titled “IntegratedDevice for Probing, Detecting and Analyzing Molecules,” which isincorporated by reference herein in its entirety.

A non-limiting example of a biological reaction taking place in areaction chamber 1-130 is depicted in FIG. 1-2, though other reactionsor analytes may be used in other applications. In this example,sequential incorporation of nucleotides or nucleotide analogs into agrowing strand 1-212 that is complementary to a target nucleic acid1-210 is taking place in the reaction chamber 1-130. The sequentialincorporation can be detected to sequence DNA. The reaction chamber mayhave a depth between about 150 nm and about 250 nm and a diameterbetween about 80 nm and about 160 nm. A metallization layer 1-240 (whichcould optionally comprise a metallization for an electrical referencepotential) may be patterned above the photodetector and provide anaperture that blocks stray light from adjacent reaction chambers andother unwanted, off-axis light sources. According to some embodiments,polymerase 1-220 may be located within the reaction chamber 1-130 (e.g.,attached to a base of the chamber). The polymerase may take up a targetnucleic acid 1-210 (e.g., a portion of nucleic acid derived from DNA),and sequence a growing strand of complementary nucleic acid to produce agrowing strand of DNA 1-212. Nucleotides or nucleotide analogs 1-310(depicted in FIG. 1-3) labeled with different fluorophores may bedispersed in a solution above the reaction chamber 1-130 and enter thereaction chamber.

When a labeled nucleotide or nucleotide analog 1-310 is incorporatedinto a growing strand of complementary nucleic acid, as depicted in FIG.1-3, one or more attached fluorophores 1-330 may be repeatedly excitedby pulses of optical energy (excitation radiation) coupled into thereaction chamber 1-130 from the waveguide 1-115. In some embodiments,the fluorophore or fluorophores 1-330 may be attached to one or morenucleotides or nucleotide analogs 1-310 with any suitable linker 1-320.An incorporation event may last for a period of time up to about 100 ms.During this time, pulses of fluorescent emission radiation resultingfrom excitation of the fluorophore(s) may be detected with sensor 1-122.By attaching fluorophores with different emission characteristics (e.g.,fluorescent decay rates, intensity, fluorescent wavelength) to thedifferent nucleotides (A,C,G,T), detecting and distinguishing thedifferent emission characteristics while each nucleic acid isincorporated into the strand of DNA 1-212 enables determination of thesequence of the growing strand of DNA. By comparing results frommultiple reaction chambers, any errors in nucleotide incorporation bythe polymerase can be detected and a genetic sequence of the target DNAcan be determined.

In some aspects, embodiments include methods of polypeptide and proteinsequencing in real-time by evaluating binding interactions of terminalamino acids with labeled amino acid recognition molecules and a labeledcleaving reagent (e.g., a labeled exopeptidase). FIG. 1-4 shows anexample of a method of sequencing in which discrete binding events giverise to signal pulses of a signal output 1-400. The inset panel of FIG.1-4 illustrates a general scheme of real-time sequencing by thisapproach. As shown, a labeled amino acid recognition molecule 1-410 canselectively bind to and dissociate from a terminal amino acid (shownhere as lysine). This binding and dissociation gives rise to a series ofpulses in signal output 1-400 that may be used to identify the terminalamino acid. In some embodiments, the series of pulses provide a pulsingpattern which may be diagnostic of the identity of the correspondingterminal amino acid.

Without wishing to be bound by theory, labeled amino acid recognitionmolecule 1-410 selectively binds and dissociates according to a bindingaffinity (K_(D)) defined by an association rate of binding (k_(on)) anda dissociation rate of binding (k_(off)). The rate constants k_(off) andk_(on) are determinants of pulse duration (e.g., the time correspondingto a detectable binding event) and interpulse duration (e.g., the timebetween detectable binding events), respectively. In some embodiments,these rates can be engineered to achieve pulse durations and pulse ratesthat give the best sequencing accuracy.

As shown in the inset panel, a sequencing reaction mixture may furthercomprise a labeled cleaving reagent 1-420 comprising a detectable labelthat is different than that of labeled amino acid recognition molecule1-410. In some embodiments, a labeled cleaving reagent 1-420 can bepresent in the mixture at a concentration that is less than that oflabeled amino acid recognition molecule 1-410. In some embodiments, alabeled cleaving reagent 1-420 displays broad specificity such that itcleaves most or all types of terminal amino acids.

As illustrated by the progress of signal output 1-400, in someembodiments, terminal amino acid cleavage by labeled cleaving reagent1-420 can give rise to a uniquely identifiable signal pulse (indicatedas a “cleavage” event in FIG. 1-4), and these events may occur withlower frequency than the binding and dissociation pulses of a labeledamino acid recognition molecule 1-410. In this way, amino acids of apolypeptide or protein can be counted and/or identified in a real-timesequencing process. As further illustrated in signal output 1-400, insome embodiments, a labeled amino acid recognition molecule 1-410 can beengineered to bind more than one type of amino acid with differentbinding and dissociation properties corresponding to each type of aminoacid, which produces uniquely identifiable pulsing patterns (e.g., asindicated by the “K,” “F,” and “Q” sets of pulses). In some embodiments,a plurality of labeled amino acid recognition molecules may be used,each with a diagnostic pulsing pattern which may be used to identify acorresponding terminal amino acid.

In some aspects, embodiments include a method of sequencing a peptide,polypeptide, or protein by detecting luminescence of a labeled peptide,polypeptide, or protein which is subjected to repeated cycles ofterminal amino acid modification and cleavage. For example, FIG. 1-5shows a method of sequencing a labeled polypeptide by Edman degradation.In some embodiments, the method generally proceeds as described forother methods of sequencing by Edman degradation. For example, in someembodiments, steps (1) and (2) shown in FIG. 1-5 may be performed forterminal amino acid modification and terminal amino acid cleavage,respectively, in an Edman degradation reaction.

As shown in the example depicted in FIG. 1-5, the method may comprise astep of (1) modifying the terminal amino acid of a labeled polypeptide.In some embodiments, modifying comprises contacting the terminal aminoacid with an isothiocyanate (e.g., PITC) to form anisothiocyanate-modified terminal amino acid 1-510. In some embodiments,an isothiocyanate modification 1-510 converts the terminal amino acid toa form that is more susceptible to removal by a cleaving reagent (e.g.,a chemical or enzymatic cleaving reagent). Accordingly, in someembodiments, the method comprises a step of (2) removing the modifiedterminal amino acid using chemical or enzymatic means for Edmandegradation.

In some embodiments, the method comprises repeating steps (1) through(2) for a plurality of cycles, during which luminescence of the labeledpolypeptide is detected. Cleavage events corresponding to the removal ofa labeled amino acid from the terminus may be detected as a decrease indetected signal, as described in connection with FIG. 1-4, for example.In some embodiments, no change in signal following step (2) as shown inFIG. 1-5 identifies an amino acid of unknown type. Accordingly, in someembodiments, partial sequence information may be determined byevaluating a signal detected following step (2) during each sequentialround by assigning an amino acid type by a determined identity based ona change in detected signal or identifying an amino acid type as unknownbased on no change in a detected signal.

Some embodiments are useful for determining amino acid sequenceinformation from peptides, polypeptides, or proteins (e.g., forsequencing one or more polypeptides). In some embodiments, amino acidsequence information can be determined for single polypeptide molecules.In some embodiments, one or more amino acids of a polypeptide arelabeled (e.g., directly or indirectly) and the relative positions of thelabeled amino acids in the polypeptide are determined. In someembodiments, the relative positions of amino acids in a protein aredetermined using a series of amino acid labeling and cleavage steps,examples of which are described above in connection with FIG. 1-4 andFIG. 1-5.

In some embodiments, the identity of a terminal amino acid (e.g., anN-terminal or a C-terminal amino acid) is assessed, after which theterminal amino acid is removed and the identity of the next amino acidat the terminus is assessed, and this process is repeated until aplurality of successive amino acids in the peptide, polypeptide, orprotein are assessed. In some embodiments, assessing the identity of anamino acid comprises determining the type of amino acid that is present.In some embodiments, determining the type of amino acid comprisesdetermining the actual amino acid identity, for example by determiningwhich of the naturally-occurring 20 amino acids is the terminal aminoacid is (e.g., using a recognition molecule that is specific for anindividual terminal amino acid). However, in some embodiments assessingthe identity of a terminal amino acid type can comprise determining asubset of potential amino acids that can be present at the terminus ofthe polypeptide. In some embodiments, this can be accomplished bydetermining that an amino acid is not one or more specific amino acids(and therefore could be any of the other amino acids). In someembodiments, this can be accomplished by determining which of aspecified subset of amino acids (e.g., based on size, charge,hydrophobicity, binding properties) could be at the terminus of thepolypeptide (e.g., using a recognition molecule that binds to aspecified subset of two or more terminal amino acids).

Amino acids of a polypeptide can be indirectly labeled, for example,using amino acid recognition molecules that selectively bind one or moretypes of amino acids on the polypeptide. Amino acids of a polypeptidecan be directly labeled, for example, by selectively modifying one ormore types of amino acid side chains on the polypeptide with uniquelyidentifiable labels. Example methods of selective labeling of amino acidside chains and details relating to the preparation and analysis oflabeled polypeptides are described in (see, e.g., Swaminathan, et al.PLoS Comput Biol. 2015, 11(2):e1004080). Accordingly, in someembodiments, the one or more types of amino acids are identified bydetecting binding of one or more amino acid recognition molecules thatselectively bind the one or more types of amino acids. In someembodiments, the one or more types of amino acids are identified bydetecting labeled polypeptide.

In some embodiments, the relative position of labeled amino acids in aprotein can be determined without removing amino acids from the proteinbut by translocating a labeled protein through a pore (e.g., a proteinchannel) and detecting a signal (e.g., a Førster resonance energytransfer (FRET) signal) from the labeled amino acid(s) duringtranslocation through the pore in order to determine the relativeposition of the labeled amino acids in the protein molecule.

As used herein, sequencing a peptide, polypeptide, or protein refers todetermining sequence information for a peptide, polypeptide, or protein.In some embodiments, this can involve determining the identity of eachsequential amino acid for a portion (or all) of the peptide,polypeptide, or protein. However, in some embodiments, this can involveassessing the identity of a subset of amino acids within the peptide,polypeptide, or protein (e.g., and determining the relative position ofone or more amino acid types without determining the identity of eachamino acid in the peptide, polypeptide, or protein). However, in someembodiments amino acid content information can be obtained from apeptide, polypeptide, or protein without directly determining therelative position of different types of amino acids in the peptide,polypeptide, or protein. The amino acid content alone may be used toinfer the identity of the peptide, polypeptide, or protein that ispresent (e.g., by comparing the amino acid content to a database ofpeptide, polypeptide, or protein information and determining whichpeptide(s), polypeptide(s), or protein(s) have the same amino acidcontent).

In some embodiments, sequence information for a plurality of polypeptideproducts obtained from a longer polypeptide or protein (e.g., viaenzymatic and/or chemical cleavage) can be analyzed to reconstruct orinfer the sequence of the longer polypeptide or protein. Accordingly, insome embodiments, the application provides compositions and methods forsequencing a polypeptide or protein by sequencing a plurality offragments of the polypeptide or protein. In some embodiments, sequencinga polypeptide or protein comprises combining sequence information for aplurality of polypeptide or protein fragments to identify and/ordetermine a sequence for the polypeptide or protein. In someembodiments, combining sequence information may be performed by computerhardware and software. The methods described herein may allow for a setof related polypeptides, such as an entire proteome of an organism, tobe sequenced. In some embodiments, a plurality ofsingle-molecule-sequencing reactions are performed in parallel (e.g., ona single bio-optoelectronic chip) according to aspects of the presentapplication. For example, in some embodiments, a plurality of singlemolecule sequencing reactions are each performed in separate samplewells on a single chip.

In some embodiments, methods provided herein may be used for thesequencing and identification of an individual protein in a samplecomprising a complex mixture of proteins. In some embodiments, theapplication provides methods of uniquely identifying an individualprotein in a complex mixture of proteins. In some embodiments, anindividual protein is detected in a mixed sample by determining at leasta partial amino acid sequence of the protein. In some embodiments, thepartial amino acid sequence of the protein is within a contiguousstretch of approximately 5 to 50 amino acids.

Without wishing to be bound by any particular theory, it is believedthat most human proteins can be identified using incomplete sequenceinformation with reference to proteomic databases. For example, simplemodeling of the human proteome has shown that approximately 98% ofproteins can be uniquely identified by detecting just four types ofamino acids within a stretch of 6 to 40 amino acids (see, e.g.,Swaminathan, et al. PLoS Comput Biol. 2015, 11(2):e1004080; and Yao, etal. Phys. Biol. 2015, 12(5):055003). Therefore, a complex mixture ofproteins can be degraded (e.g., chemically degraded, enzymaticallydegraded) into short polypeptide fragments of approximately 6 to 40amino acids, and sequencing of this polypeptide library would reveal theidentity and abundance of each of the proteins present in the originalcomplex mixture. Compositions and methods for selective amino acidlabeling and identifying polypeptides by determining partial sequenceinformation are described in detail in U.S. patent application Ser. No.15/510,962, filed Sep. 15, 2015, titled “SINGLE MOLECULE PEPTIDESEQUENCING,” which is incorporated by reference in its entirety.

Sequencing in accordance with the application, in some aspects, caninvolve immobilizing a peptide, polypeptide, or protein on a surface ofa substrate or solid support, such as a chip or integrated device. Insome embodiments, a peptide, polypeptide, or protein can be immobilizedon a surface of a sample well (e.g., on a bottom surface of a samplewell) on a substrate. In some embodiments, a first terminus of apeptide, polypeptide, or protein is immobilized to a surface, and theother terminus is subjected to a sequencing reaction. For example, insome embodiments, a polypeptide is immobilized to a surface through aC-terminal end, and terminal amino acid recognition and degradationproceeds from an N-terminal end of the polypeptide toward the C-terminalend. In some embodiments, the N-terminal amino acid of the polypeptideis immobilized (e.g., attached to the surface). In some embodiments, theC-terminal amino acid of the polypeptide is immobilized (e.g., attachedto the surface). In some embodiments, one or more non-terminal aminoacids are immobilized (e.g., attached to the surface). The immobilizedamino acid(s) can be attached using any suitable covalent ornon-covalent linkage. In some embodiments, a plurality of peptides,polypeptides, or proteins are attached to a plurality of sample wells orreaction chambers of a bio-optoelectronic chip or integrated devicedescribed in connection with FIG. 1-1 and FIG. 1-2 (e.g., with onepeptide, polypeptide, or protein attached to a surface, for example abottom surface, of each sample well).

Optical microdisks described herein that can improve collection ofemission radiation are not limited to only applications in instrumentsconfigured for genetic or polypeptide sequencing or to use only inconnection with integrated devices having the structure described inFIG. 1-1 and FIG. 1-2. More generally, embodiments of optical microdisksdescribed herein may be used in applications in which it is desired toincrease SNR or increase a desired light intensity by increasing thecollection of emission radiation or other radiation for microscaledevices. Among other possible contexts, optical microdisks describedherein may be used in conjunction with, for example, integrateddetectors in optical communication systems (improved signal collection),imaging arrays (improved signal collection), and/or LED emitters oremitting arrays (improved concentration of emission).

Referring to FIG. 1-6, in some implementations a microdisk 1-605 may bedisposed within at least one surrounding medium 1-610 between thewaveguide 1-112 and the sensor 1-122. According to some embodiments, themicrodisk 1-605 may be made of one or more materials transparent at thewavelength of the emission radiation, and with a refractive indexdifferent (e.g., greater) than the refractive index of the surroundingmedium 1-610. As a non-limiting example, the microdisk may be formed ofsilicon nitride, and the surrounding medium 1-610 may be formed ofsilicon dioxide. A dielectric material that may be used to form themicrodisk 1-605 may be amorphous, mono-crystalline, or poly-crystalline,doped or undoped, and/or an alloy of two or more materials. Otherexample materials include, but are not limited to aluminum oxide,titanium nitride, titanium oxide, tantalum nitride, and tantalum oxide.In some embodiments, the material from which a microdisk is made may betransparent to a characteristic wavelength of the emission radiation(e.g., transmit at least 80% of the intensity at the characteristicwavelength). In some cases, the material from which a microdisk is mademay be semi-transparent to a characteristic wavelength of the emissionradiation (e.g., transmit between 50% and 80% of the intensity at thecharacteristic wavelength). By having a higher refractive index than thesurrounding medium, the microdisk 1-415 can effectively collect andconcentrate emission radiation from the reaction chamber 1-130 andre-radiate the emission in a concentrated manner onto a correspondingsensor 1-122 as compared to a same structure that does not have amicrodisk.

According to some embodiments, a microdisk comprises a resonant cavity.In some cases, the resonant cavity may be a weak resonant cavity (e.g.,an optical cavity having a quality (Q) factor between 10 and 100 orbetween 10 and 1000). The resonant cavity is capable of collectingemission radiated from the reaction chamber 1-130 and re-radiating theemission with improved directionality toward the sensor 1-122. “Improveddirectionality” in this context means that the re-radiated emission iscondensed and directed toward the sensor 1-122 compared to a case whenthe microdisk 1-605 is not present. For example, a transverse intensitybeam profile (FWHM value) of a beam of re-radiated emission travellingfrom the microdisk 1-605 to the sensor 1-122 is less than a transverseintensity beam profile (FWHM value) of a beam of radiated emissiontravelling from the reaction chamber to the sensor 1-122 when themicrodisk is not present, where both beam profiles are measured at asame location (e.g., at an entrance surface to the sensor 1-122). Byreducing the transverse intensity beam profile, more radiation can becondensed onto the sensor 1-122. In some cases, the reduction intransverse intensity beam profile (FWHM) is between 10% and 50%.

In some embodiments, the microdisk 1-605 may be shaped as a circulardisk having a thickness t and diameter D, thereby providing rotationalsymmetry. In some embodiments, the microdisk 1-605 may be shaped as anellipse, a hexagon, an octagon, a square, a triangle, or any othersuitable shape. In some cases, a microdisk 1-605 may be positioned suchthat the center of the disk is essentially aligned along a z-axis thatruns through a center of the reaction chamber 1-130. In someembodiments, reaction chamber 1-130, microdisk 1-605, and sensor 1-122may be aligned to one another along the z-axis.

The phrase “characteristic wavelength” or “wavelength” is used to referto a central or predominant wavelength within a limited bandwidth ofradiation (e.g., a central or peak wavelength within an excitationbandwidth for excitation radiation or within an emission bandwidth foremission radiation). In some cases, “characteristic wavelength” or“wavelength” may be used to refer to a peak wavelength within a totalbandwidth of radiation output by a source.

In some implementations, a microdisk 1-605 may be disposed between 500nm and 1500 nm below the waveguide 1-115 or an optical source. In somecases, a microdisk 1-605 may be disposed between 800 nm and 1300 nmbelow the waveguide 1-115 or an optical source. In some implementations,a microdisk 1-605 may be disposed between 900 and 1250 nm below thewaveguide 1-115 or an optical source. Improved performance may beobtained when a microdisk 1-605 is disposed between 1000 and 1500 nmbelow the waveguide 1-115 or an optical source. Further, in someembodiments, a microdisk 1-605 may have a thickness t along the z-axisof between 100 nm and 800 nm and a diameter D between 0.5 microns (μm)and 2 μm.

According to some embodiments, a microdisk 1-605 may be surrounded byone or more radially symmetric rings 1-705, as depicted in FIG. 1-7.Rings 1-705 may be formed of the same material or a different materialas microdisk 1-605, such as silicon nitride or any other materialdescribed above that is used to form the microdisk. In someimplementations, the one or more rings 1-705 may be formed during a sameprocess that is used to form the microdisk 1-605 and may be concentricwith a central vertical axis of the microdisk 1-605. According to someembodiments, there may be only one ring surrounding a microdisk 1-605.The one or more rings 1-705 may be formed at a same level as themicrodisk or may be offset in the z-direction. The one or more rings1-705 may further increase an amount of emission radiation received atthe sensor 1-122, compared to a microdisk 1-605 only. For example, theone or more rings 1-705 and microdisk 1-605 may be patterned andarranged as a Fresnel zone plate (or approximation thereof) for acharacteristic emission wavelength having a value between 560 nm and 700nm. When present, the one or more rings may provide furtherconcentration, focusing, and/or improved directionality of the emissionre-radiated onto the sensor 1-122 compared to a microdisk alone. In somecases when one or more rings 1-705 are used, the reduction in transverseintensity beam profile (FWHM) may be between 20% and 70%.

According to some embodiments, the rings 1-705 and intervening medium1-610 provide regions of alternating optical material. For example, therings 1-705 and intervening medium 1-610 may alternate between regionsof a first refractive index and a second refractive index and/or regionsof a first optical transmissivity and regions of a second opticaltransmissivity for the emission radiation. The alternating regions maycause diffraction such that radiation transmitted through the moretransparent regions to constructively interfere at a desired focalpoint, e.g., a center of the sensor 1-122, for the characteristicemission wavelength. In some cases, enhancement of emission radiationreceived at the sensor 1-122 may occur even if rings 1-705 are notconfigured as a Fresnel zone plate or even if only one ring 1-705 ispresent in the integrated device.

A thickness of the one or more rings 1-705 may be essentially the sameas or different from a thickness of the microdisk. In someimplementations, a thickness of the one or more rings 1-705 along thez-axis can be any value between 100 nm and 800 nm. A diameter of a ringcan be any value between 0.6 μm and 4 μm. A size of a gap between rings1-705 along the x-axis may vary within a device and can be any valuebetween 100 nm and 500 nm.

Another example of optical structures that may be included at a pixel ofan integrated device is shown in FIG. 2-1. According to someimplementations, one or more iris layers 2-125 may be formed above thesensor 1-122. An iris layer 2-125 may include an opening or hole 2-112through a light-reducing material. The light-reducing material maycomprise a metal, polymer, semiconductor, or any material that rejects(e.g., absorbs and/or reflects) a majority of excitation radiationincident on the iris layer 2-125. The light-reducing material may alsoreject emission radiation in some cases. The hole 2-112 can allowemission from the reaction chamber 1-130 to pass through the iris layer2-125 and reach the sensor 1-122, while the light-reducing materialblocks or attenuates radiation from other directions (e.g., fromadjacent pixels or from scattered excitation radiation). For example, aniris layer 2-125 can block or attenuate scattered excitation radiationat wide angles of incidence from striking the sensor 1-122 andcontributing to background noise. In some embodiments, an iris layer2-125 may be formed from a conductive material and provide a potentialreference plane or grounding plane for circuitry formed on or above thesubstrate 1-105. In some embodiments, an iris layer 2-125 may be formedfrom a dielectric material. The hole 2-112 in the iris layer may beshaped in any suitable way, such as a square, rectangle, disk, ellipse,polygon, etc.

In the example of FIG. 2-1, two iris layers 2-125 are included. One irislayer is disposed between the microdisk 1-605 and a discriminatingoptical structure 2-120 that may be configured to pass emissionradiation and attenuate excitation radiation that is incident on thesensor 1-122. One iris layer is disposed between the discriminatingoptical structure 2-120 and a complementary metal-oxide-semiconductor(CMOS) circuitry 2-110 and/or interconnects. Examples of adiscriminating optical structure 2-120 include, but are not limited to,a diffraction grating filter, a multi-layer dielectric optical filter, asingle or multi-layer semiconductor absorber exhibiting a band edge (asdescribed in U.S. Provisional Application Ser. No. 62/831,237 filed onApr. 9, 2019, titled “Semiconductor Optical Absorption Filter for anIntegrated Device” which is incorporated by reference herein in itsentirety), and a microfabricated structure having periodic orquasi-periodic modulations in refractive index in two orthree-dimensions such as a photonic band-gap structure (as described inU.S. Provisional Application Ser. No. 62/863,635 filed on Jun. 19, 2019,titled “Optical Nanostructure Rejecter for an Integrated Device andRelated Methods,” which is incorporated by reference herein in itsentirety. Although two iris layers 2-125 are shown in FIG. 2-1, theremay be fewer or more iris layers at a pixel of an integrated device. Insome cases, a single iris layer may be used and may be located betweenthe reaction chamber 1-130 and microdisk 1-605 or between the microdisk1-605 and sensor 1-122. In some embodiments, there may be three or moreiris layers located between a waveguide 1-115 and sensor 1-122. Theopening diameters of the irises, when multiple irises are used, may bethe same or different. In some embodiments, one or more interconnectlayers with CMOS circuitry 2-110 may be patterned to form an iris for asensor 1-122.

In some implementations, there can be one or more additional transparentor semitransparent layers 2-130 formed on the substrate 1-105 andlocated between the substrate and the optical waveguide 1-115. Theseadditional layers may be formed from an oxide or a nitride, and may beof the same type of material as the transparent or semitransparentmaterial that the reaction chamber 1-130 is formed in or as thesurrounding medium 1-610 that the microdisk 1-605 is formed in,according to some embodiments.

According to some embodiments, a microdisk 1-605 comprises amicro-resonator that can couple in emission radiation from the reactionchamber 1-130 and re-emit the radiation towards the sensor 1-122. Insome implementations, the microdisk 1-605 may efficiently coupleemission radiation traveling at an angle to the vertical z axis intoresonant optical modes of the microdisk and re-radiate the coupledemission towards the sensor 1-122, thereby improving collection of theseoff-axis emissions from the reaction chamber 1-130. To enhance resonatorcharacteristics, a thickness t of the microdisk 1-605, divided by arefractive index of the microdisk, may be an integral number of halfwavelengths. According to some example embodiments, a thickness t of asilicon nitride microdisk may be approximately 200 nm, approximately 350nm, or approximately 480 nm for emission radiation having acharacteristic wavelength of about 570 nm. The inventors have found thatmicrodisks having a thickness of 400 nm or more provide bettercollection of emission radiation than microdisks having a thickness lessthan 300 nm.

FIGS. 2-2 and 2-3 depict example optical intensity calculated for apixel of an integrated device having a structure similar to thatdepicted in FIG. 2-1. For this simulation, the waveguide 1-115 andmicrodisk 1-605 comprise silicon nitride surrounded by silicon oxide.Two irises 2-125 are located between the microdisk 1-605 and CMOS layers(one shown) 2-110. The microdisk 1-605 is formed as a circular disk. Forthis simulation, the microdisk 1-605 has a thickness of 450 nm andradius of 1.2 μm, and the top of the microdisk is located approximately1.4 μm below waveguide 1-115. The two irises each have a diameter of 1.6μm and are separated vertically by approximately 1 μm. In some cases,the irises may be separated vertically by a distance between 0.5 μm and3 μm. A sensor may be located just below the CMOS circuitry 2-110,though is not shown in the plots. For this example simulation, theexcitation radiation has a characteristic wavelength (λ=λ_(excitation))of 532 nm and the emission radiation has a characteristic wavelength(λ=λ_(emission)) of 572 nm. The optical intensity patterns in FIG. 2-2and FIG. 2-3 were computed with software that solves Maxwell's equations(e.g., using a finite-difference time-domain analysis) within asimulation domain. In this example, the following initial conditionswere used for the excitation and emission radiation: 1) radiation atλ=λ_(excitation) is coupled into the single-mode waveguide 1-115 from anexternal source and illuminates the waveguide 1-115 uniformly along thelength of the waveguide, and 2) radiation at λ=λ_(emission) is generatedin the reaction chamber 1-130 in response to the excitation radiation.It will be appreciated that the parameters given above in connectionwith FIG. 2-2 and FIG. 2-3 are for illustrative purposes only, and thatother wavelengths and other optical nanostructure parameters(periodicity, width, thickness, etc.) may be used.

As illustrated in FIG. 2-2, for λ=λ_(emission), a significant portion ofthe emission radiation is collected by microdisk 1-605 and guidedthrough irises 2-125 toward the sensor 1-122. Such an increase inradiation collection may increase the SNR, resulting in faster and/ormore accurate measurements. In FIG. 2-2, the reaction chamber 1-130 andwaveguide 1-115 are aligned so that their centers are directly overcenters of the microdisk 1-605 and apertures 2-125. Accordingly, theemission radiation travelling from the microdisk 1-605 may fallcentrally on a sensor 1-122 located below the irises 2-125.

The inventors have also recognized and appreciated that semiconductorfabrication requires aligning multiple layers during the fabricationprocess, and that misalignment of layers may occur. In FIG. 2-3, thereaction chamber 1-130 and waveguide 1-115 are aligned so that theircenters are shifted laterally by about 250 nm from the centers of themicrodisk 1-605 and irises 2-125. Even for such a misalignment, themicrodisk 1-605 can still collect and guide a significant portion of theemission radiation towards the sensor 1-122. The sensor 1-122 may have adetection area that is larger than the lower iris 2-125, and theemission radiation may fall off-center on the sensor 1-122. Collectionof emission radiation by the microdisk 1-605 may therefore toleratemisalignment (e.g., up to at 250 nm or more) of components infabrication of such integrated devices.

For a pixel of an integrated device such as the example depicted in FIG.1-4, FIG. 1-5, or FIG. 2-1, the amount of emission radiation collectedby the sensor will typically depend upon one or more physical parametersof the structure (e.g., microdisk thickness, microdisk diameter,microdisk material, surrounding medium material, iris location, irisdiameter, distance of microdisk from the reaction chamber, etc.). One ormore of these parameters can be selected and/or adjusted formicrofabrication to improve performance of the optical detection withina pixel and increase an amount of emission radiation received by asensor 1-122. For example, increasing a thickness of the microdisk 1-605and/or changing its spacing from the reaction chamber 1-130 may increasean amount of emission radiation received by a sensor 1-122 within thepixel. Additionally or alternatively, changing iris diameters and/orlocations of irises may increase an amount of emission radiationreceived by a sensor 1-122 within the pixel.

FIG. 3-1 is a plot of simulation results illustrating normalizedcollection efficiency (vertical axis) plotted as a function of verticaldistance (horizontal axis) between the microdisk 1-605 and a metal layercoating 1-150 surrounding a reaction chamber 1-130. The simulationresults are for a pixel structure such as that of FIG. 2-1 (thoughwithout a discriminating optical structure 2-120). The collectionefficiency is an amount of intensity received at a sensor 1-122normalized to the highest amount of intensity received by the sensor1-122 over the range of distances used in the simulation. The emissionradiation has a characteristic wavelength of 572 nm in this example.

In FIG. 3-1, the normalized collection efficiency is plotted for adistance between the microdisk 1-605 and the coating 1-150 which variesbetween 1200 nm and 1775 nm. The distance is measured from a top of themetal coating 1-150 to a top of the microdisk 1-605. The normalizedcollection efficiency exhibits a periodic behavior with a period ofapproximately 200 nm. The normalized collection efficiency furtherexhibits an average slope which decreases as the distance between themicrodisk 1-605 and the metal coating 1-150 increases. The periodicbehavior is associated with the resonant characteristics of a microdisk1-605, as described above. The periodic behavior can have a furtherdependence on the refractive index of the microdisk 1-605 and arefractive index of material(s) surrounding the microdisk and/orwaveguide. The surrounding material (oxide in this example) may bereferred to as cladding material. The periodic behavior indicates thatthere are preferred locations (e.g., at or near the maxima of the curve)for which the microdisk 1-605 within a pixel of an integrated devicewill provide improved collection efficiency. In some embodiments, thepreferred locations may correspond to a distance between a top of themicrodisk 1-605 and the metal coating 1-150 being approximately equal toan integer number of half wavelengths of the emission radiation in thecladding material.

Improved collection efficiency and an increase in an amount of signalreceived by a sensor is indicated in the plots of FIG. 3-2, FIG. 3-3,and FIG. 3-4 for example simulations. The results plotted in FIG. 3-2are for a same overall structure like that shown in FIG. 2-1 (withoutthe discriminating optical structure 2-120), but for two differentcases. The lower curve 3-220 is for a pixel structure that does notinclude a microdisk. The upper curve 3-210 is for pixel structure thatincludes a microdisk 1-605. FIG. 3-2 plots simulated collectionefficiency (vertical axis) as a function of iris diameter (horizontalaxis) for each case. The upper curve 3-210 (structure with microdisk)shows a higher collection efficiency than the lower curve 3-220(structure without microdisk) for all iris diameters. For thesesimulations, collection efficiencies plotted are a ratio of intensitypassed through the last iris before the sensor 1-122 to the totalintensity of emission radiation from the reaction chamber 1-130. For theexample pixel structures of the simulations, two irises were locatedbetween the waveguide 1-115 and sensor 1-122. The upper iris was located2 microns from the waveguide and 1.7 microns from the reaction chamber1-130. The irises were spaced 2.5 microns apart. The surrounding medium1-610 around the waveguide and irises was silicon oxide. For the casewith the microdisk, the microdisk had a diameter of 1400 nm and athickness of 480 nm. A top of the microdisk was spaced 1 micron from thebottom of the reaction chamber 1-130.

Although collection efficiency increases with iris diameter, larger irisdiameters may pass more unwanted radiation (e.g., scattered excitationradiation) to the sensor 1-122. Therefore, it can be beneficial to usesmaller iris diameters (e.g., diameters less than 2.5 microns). For someiris diameters, the collection efficiency with a microdisk can bebetween 2 and 5 times the collection efficiency without a microdisk(e.g., with iris diameters in a range between 1 micron and 3 microns).

The results shown in FIG. 3-3 indicate that there can be a preferredpairing of microdisk diameter and iris diameters. The conditions used togenerate the data for FIG. 3-3 were the same as those used to generatethe data for FIG. 3-2, however microdisk and iris diameters were varied.The plot shows contours of collection efficiency normalized to thehighest value of collection efficiency obtained over the range of valuesused to generate the plot. The plot may be used to select a diameter fora microdisk 1-605 if iris diameters have been determined or areconstrained. For example, if iris diameters are selected to beapproximately 1.2 microns for purposes of blocking a desired amount ofexcitation radiation, then a microdisk having a diameter ofapproximately 1 micron would provide a higher collection efficiency thana microdisk having a diameter of approximately 1.2 micron.

The results plotted in FIG. 3-4 show how changes in upper and lower irisdiameters can affect an increase in an amount of emission radiationreceived by a sensor 1-122. The conditions used to generate the data forFIG. 3-4 were the same as those used to generate the data for FIG. 3-2,however the iris diameters were varied independently. The plot showscontours of an enhancement factor (a ratio of an amount of emissionradiation received by the sensor with the microdisk to an amount ofemission radiation received by the sensor without the microdiskpresent).

Two example microfabricated structures that include microdisks and thatmay be used in an integrated device are shown in the scanning electronmicroscopy (SEM) images of FIG. 4-1 and FIG. 4-2. Several physicalparameters are different in the two example structures. Parameters thatcan be adjusted controllably during microfabrication include thedistances dl between the bottom surface of the waveguides 4-115 and4-215 (or reaction chambers 4-130, 4-230) and the top surface of themicrodisks 4-105 and 4-205, respectively. The diameters and thicknessesof the microdisks 4-105 and 4-205 can also be adjusted controllably, asdepicted. Additionally, the diameters of the openings of irises 4-125and 4-225 and their locations can be adjusted controllably as depicted.In FIG. 4-1, the microdisk 4-105 has a larger diameter than the openingof iris 4-125, while in FIG. 4-2 the microdisk 4-205 has a smallerdiameter than the opening of iris 4-225. In these examples, multi-layeroptical filters 4-120, 4-220 are located below the irises 4-125, 4-225.Other parameters that may affect the collection efficiency include thevertical distance between the microdisks 4-105, 4-205 and the irislayers 4-125, 4-225, which can be controllably adjusted duringmicrofabrication.

II. Methods for Fabricating Optical Microdisks

FIGS. 5-1A through 5-1F illustrate example structures associated withmicrofabrication steps that may be used to form an optical microdisk ata pixel of an integrated device. Although structure for only one pixelis shown, it will be appreciated that multiple pixels can be fabricatedsimultaneously using planar microfabrication processes in accordancewith the illustrated embodiments. In FIG. 5-1A, a substrate 5-105 may beprovided or obtained upon which lithography steps may be performed.Substrate 5-105 may include some structure already formed on thesubstrate 5-105. For example, substrate 5-105 may include part of thestructure shown in FIG. 1-1 or FIG. 2-1 below the microdisk 5-405, suchas iris 5-125 and/or CMOS circuitry. In some embodiments, substrate5-105 may comprise a bulk semiconductor substrate, though other types ofbulk substrates may be used in some implementations. In the example ofFIG. 5-1A, substrate 5-105 includes an iris layer 5-125 and a planarizedlayer of silicon dioxide 5-130 above the iris layer 5-125.

According to some embodiments, a first material layer 5-110 may bedeposited or grown on substrate 5-105, as depicted in FIG. 5-1B. Thefirst material layer 5-110 may comprise a high index dielectric materialsuch as silicon nitride, and may be deposited to a thickness equal tothe desired thickness of resulting microdisk 5-405. The first materiallayer 5-110 may be deposited, for example, by physical vapor deposition(PVD) techniques such as sputtering or chemical vapor deposition (CVD)techniques such as plasma-enhanced chemical vapor deposition (PECVD) orhigh-density plasma (HDP) PECVD.

Subsequently, a photoresist layer may be deposited over first materiallayer 5-110 and then patterned (not shown). The patterned photoresistlayer may be used as an etch mask to etch the first material layer 5-110into a desired pattern, depicted in FIG. 5-1C. Etching may be done by,for example, a wet etching process or a plasma etching process such as areactive ion etch (RIE) or a deep reactive ion etch (DRIE). Theremaining photoresist may be removed in a cleaning step, leaving apatterned structure such as the structure depicted in FIG. 5-1C. Theresulting structure of the etched first material layer 5-110 may includea microdisk 5-405 and a plurality of residual structures 5-112 separatedfrom the microdisk and formed from a same material as the microdisk5-405. In some cases, the residual structures 5-112 may be removed bymasking the microdisk 5-405 and etching away the residual structures. Insome embodiments, the residual structures may be retained, and mayimprove fidelity of the microdisk by providing, for example, anetch-stop material for a subsequent planarization step.

According to some implementations, an overcoat layer 5-120 can then bedeposited to fill voids 5-115 and cover the microdisk structure 5-405and the residual structures 5-112. The overcoat layer 5-120 may bedeposited by any suitable method such as PVD, PECVD, HDP PECVD, orsputtering. The overcoat layer 5-120 may be formed of any suitablematerial such as silicon dioxide, as a non-limiting example. In somecases, due to the structure of voids 5-115, overcoat layer 5-120 may notbe able to form a smooth top surface. Overcoat layer 5-120 may then beplanarized by, for example, chemical-mechanical polishing (CMP) to forma planar surface 5-122 for subsequent processing, as depicted in FIG.5-1E.

Optionally, one or more additional material layers 5-130 may bedeposited onto the structure of FIG. 5-1E to form the structure of FIG.5-1F. Additional layer(s) 5-130 may be deposited by any suitable methodsuch as PVD, PECVD, HDP PECVD, or sputtering. The additional layers(s)5-130 may be formed of any suitable material such as silicon dioxide, asa non-limiting example. Additional layers(s) 5-130 may be the samematerial as overcoat layer 5-120 or may be a different material. In someembodiments, additional layer(s) 5-130 may be planarized by CMP,additionally or alternatively to planarization of overcoat layer 5-120,to provide a smooth surface free of unwanted voids. Such a surface canbe beneficial for low-loss waveguides such as those described herein.After the fabrication of the microdisk structure of FIG. 5-1F,additional components such as the waveguide and reaction chamber may befurther fabricated on top of the structure to form a pixel of anintegrated device, such as shown in the examples of FIG. 1-1 and FIG.2-1.

In some embodiments and referring again to FIG. 5-1D, the overcoat layer5-120 may not be planarized. The structure of the overcoat layer 5-120over the microdisk 5-405 can exhibit some positive lensing near theedges of the microdisk 5-405, provided the immediately additional layer5-130 has a lower refractive index. According to some embodiments, theimmediately additional layer 5-130 may fill voids in the overcoat layer5-120, have a lower index of refraction than the overcoat layer 5-120 ata characteristic wavelength of the emission radiation, and have aplanarized top surface. Such structure may further increase collectionefficiency of emission radiation.

III. Conclusion

Having thus described several aspects of several embodiments of anoptical microdisk, it is to be appreciated that various alterations,modifications, and improvements will readily occur to those skilled inthe art. Such alterations, modifications, and improvements are intendedto be part of this disclosure, and are intended to be within the spiritand scope of the invention. While the present teachings have beendescribed in conjunction with various embodiments and examples, it isnot intended that the present teachings be limited to such embodimentsor examples. On the contrary, the present teachings encompass variousalternatives, modifications, and equivalents, as will be appreciated bythose of skill in the art.

While various inventive embodiments have been described and illustrated,those of ordinary skill in the art will readily envision a variety ofother means and/or structures for performing the function and/orobtaining the results and/or one or more of the advantages described,and each of such variations and/or modifications is deemed to be withinthe scope of the inventive embodiments described. More generally, thoseskilled in the art will readily appreciate that all parameters,dimensions, materials, and configurations described are meant to beexamples and that the actual parameters, dimensions, materials, and/orconfigurations will depend upon the specific application or applicationsfor which the inventive teachings is/are used. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific inventive embodimentsdescribed. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, inventiveembodiments may be practiced otherwise than as specifically describedand claimed. Inventive embodiments of the present disclosure may bedirected to each individual feature, system, system upgrade, and/ormethod described. In addition, any combination of two or more suchfeatures, systems, and/or methods, if such features, systems, systemupgrade, and/or methods are not mutually inconsistent, is includedwithin the inventive scope of the present disclosure.

Further, though some advantages of the present invention may beindicated, it should be appreciated that not every embodiment of theinvention will include every described advantage. Some embodiments maynot implement any features described as advantageous. Accordingly, theforegoing description and drawings are by way of example only.

All literature and similar material cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, and web pages, regardless of the format of suchliterature and similar materials, are expressly incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The section headings used are for organizational purposes only and arenot to be construed as limiting the subject matter described in any way.

Also, the technology described may be embodied as a method, of which atleast one example has been provided. The acts performed as part of themethod may be ordered in any suitable way. Accordingly, embodiments maybe constructed in which acts are performed in an order different thanillustrated, which may include performing some acts simultaneously, eventhough shown as sequential acts in illustrative embodiments.

All definitions, as defined and used, should be understood to controlover dictionary definitions, definitions in documents incorporated byreference, and/or ordinary meanings of the defined terms.

Numerical values and ranges may be described in the specification andclaims as approximate or exact values or ranges. For example, in somecases the terms “about,” “approximately,” and “substantially” may beused in reference to a value. Such references are intended to encompassthe referenced value as well as plus and minus reasonable variations ofthe value. For example, a phrase “between about 10 and about 20” isintended to mean “between exactly 10 and exactly 20” in someembodiments, as well as “between 10±δ1 and 20±δ2” in some embodiments.The amount of variation δ1, δ2 for a value may be less than 5% of thevalue in some embodiments, less than 10% of the value in someembodiments, and yet less than 20% of the value in some embodiments. Inembodiments where a large range of values is given, e.g., a rangeincluding two or more orders of magnitude, the amount of variation δ1,δ2 for a value could be as high as 50%. For example, if an operablerange extends from 2 to 200, “approximately 80” may encompass valuesbetween 40 and 120 and the range may be as large as between 1 and 300.When exact values are intended, the term “exactly” is used, e.g.,“between exactly 2 and exactly 200.”

The term “adjacent” may refer to two elements arranged within closeproximity to one another (e.g., within a distance that is less thanabout one-fifth of a transverse or vertical dimension of a larger of thetwo elements). In some cases there may be intervening structures orlayers between adjacent elements. In some cases adjacent elements may beimmediately adjacent to one another with no intervening structures orelements.

The indefinite articles “a” and “an,” as used in the specification andin the claims, unless clearly indicated to the contrary, should beunderstood to mean “at least one.”

The phrase “and/or,” as used in the specification and in the claims,should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used shall only be interpreted as indicating exclusive alternatives(i.e., “one or the other but not both”) when preceded by terms ofexclusivity, such as “either,” “one of,” “only one of,” or “exactly oneof.” “Consisting essentially of,” when used in the claims, shall haveits ordinary meaning as used in the field of patent law.

As used in the specification and in the claims, the phrase “at leastone,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively.

The claims should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail may be made by one of ordinary skillin the art without departing from the spirit and scope of the appendedclaims. All embodiments that come within the spirit and scope of thefollowing claims and equivalents thereto are claimed.

What is claimed is:
 1. A microfabricated optical structure, comprising:a substrate having a plurality of pixels wherein two or more of theplurality of pixels each comprise: a reaction chamber configured to holda specimen for analysis; a waveguide configured to deliver excitationradiation to the reaction chamber; an optical sensor configured todetect emission radiation emitted from the reaction chamber; and amicrodisk disposed between the waveguide and the optical sensor andconfigured to increase an amount of the emission radiation that isreceived by the optical sensor compared to an amount of the emissionradiation that would be received by the optical sensor without themicrodisk.
 2. The microfabricated structure of claim 1, wherein themicrodisk forms a resonant cavity for collecting and re-radiating theemission radiation.
 3. The microfabricated structure of claim 1, whereinthe microdisk is surrounded by at least one concentric ring configuredincrease the amount of the emission radiation that is received by theoptical sensor.
 4. The microfabricated structure of claim 3, wherein theat least one concentric ring comprises a same material as the microdisk.5. The microfabricated structure of claim 1, wherein the microdiskcomprises a dielectric material having a first index of refraction thatis different from a second index of refraction for material surroundingthe microdisk.
 6. The microfabricated structure of claim 1, wherein themicrodisk comprises an oxide or nitride material.
 7. The microfabricatedstructure of claim 1, wherein the microdisk is formed from siliconnitride.
 8. The microfabricated structure of claim 1, wherein themicrodisk is disposed between 500 nm and 1500 nm below the waveguide. 9.The microfabricated structure of claim 1, wherein the microdisk has athickness between 100 nm and 800 nm.
 10. The microfabricated structureof claim 1, wherein the microdisk has an elliptical cross-section takenin a plane parallel to a surface of the substrate.
 11. Themicrofabricated structure of claim 1, wherein the microdisk has acircular cross-section taken in a plane parallel to a surface of thesubstrate.
 12. The microfabricated structure of claim 11, wherein themicrodisk has a diameter between 0.5 um and 2 um.
 13. Themicrofabricated structure of claim 1, further comprising an opticalfilter disposed below the microdisk that is configured to reduceexcitation radiation incident on the optical sensor.
 14. Themicrofabricated structure of claim 13, wherein the optical filtercomprises a microfabricated structure having periodic modulations inrefractive index values in two or three dimensions.
 15. Themicrofabricated structure of claim 1, further comprising at least oneiris layer disposed below the microdisk and configured to allow theemission radiation from the reaction chamber to reach the optical sensorwhile blocking at least some scattered excitation radiation fromreaching the optical sensor.
 16. The microfabricated structure of claim1, further comprising complementary metal-oxide-semiconductor (CMOS)circuitry integrated on the substrate and connected to the opticalsensor.
 17. The microfabricated structure of claim 1, wherein thewaveguide is arranged to deliver excitation radiation to a plurality ofthe pixels.
 18. A method of operating an integrated device, the methodcomprising: delivering excitation radiation from an optical waveguide toa reaction chamber, wherein the optical waveguide and reaction chamberare integrated on a substrate of the integrated device; passing emissionradiation from the reaction chamber through a microdisk to a sensor thatis integrated on the substrate; and increasing with the microdisk anamount of the emission radiation received by the sensor compared to anamount of the emission radiation that would be received without themicrodisk.
 19. The method of claim 18, further comprising passing theemission radiation from the reaction chamber through an iris.
 20. Themethod of claim 19, further comprising blocking, at least in part, theexcitation radiation with the iris.
 21. The method of claim 18, furthercomprising passing the emission radiation from the reaction chamberthrough a discriminating optical structure.
 22. The method of claim 18,wherein the microdisk comprises a dielectric material having a firstindex of refraction that is different from a second index of refractionfor material surrounding the microdisk.
 23. The method of claim 18,wherein the microdisk comprises an oxide or nitride material.
 24. Themethod of claim 23, wherein the microdisk is formed from siliconnitride.
 25. A method for fabricating an integrated device, the methodcomprising: forming, at each of a plurality of pixels on a substrate, areaction chamber, an optical waveguide arranged to deliver excitationradiation to the reaction chamber, and an optical sensor arranged toreceive emission radiation from the reaction chamber; and forming amicrodisk at each pixel between the optical waveguide and the opticalsensor, wherein the microdisk is configured to increase an amount of theemission radiation that is received by the optical sensor compared to anamount of the emission radiation that would be received without themicrodisk.
 26. The method of claim 25, wherein forming the microdiskcomprises depositing a first dielectric material onto the substrate andetching said first dielectric material to form voids in the firstdielectric material.
 27. The method of claim 26, wherein forming themicrodisk further comprises filling the voids in the first dielectricmaterial with a second material different than the first dielectricmaterial.
 28. The method of claim 27, wherein the first dielectricmaterial has a first index of refraction and the second material has asecond index of refraction different from the first index of refraction.29. The method of claim 26, wherein the first dielectric material issilicon nitride.
 30. The method of claim 25, further comprising formingat least one iris layer prior to forming the microdisk at each pixel.31. The method of claim 25, further comprising forming an optical filterprior to forming the microdisk at each pixel.
 32. The method of claim25, further comprising performing a planarization process prior toforming the optical waveguide at each pixel.